At this writing, the committee is not aware of any existing nucleic acid analysis system that performs the full set of tasks described above for relevant aerosol concentrations of pathogenic organisms within the detect-to-warn time constraint of 1 minute. Presently, this type of multistep analysis is performed manually in laboratory settings by highly trained personnel and requires an hour or more to accomplish. Recent advances in the miniaturization of instrumentation, however, offer the possibility that such nucleic acid-based analysis protocols could be automated and performed in the field by dedicated stand-alone sensors, given much additional development.

This chapter describes representative present-day technologies that perform each of the functions listed above (in sequential order, as a sensor system would operate) and evaluates their potential for use in detect-to-warn applications. Aside from technological feasibility, other factors must also be considered: e.g., sensitivity, specificity, robustness to environmental contaminants, requirements for sample preparation, cost, storage and logistics, ease of implementation into field equipment, and level of multiplexing of the assays. The chapter concludes with the committee's key findings and recommendations.

SAMPLE COLLECTION

As with any other sensor methodology discussed in this report, a nucleic acid sensor would first need to collect an air sample of sufficient volume and pathogen density to be detectable in the assay. For most of the techniques described below, the assay is performed with the organism and target nucleic acid sequence in aqueous solution. Therefore, the air-collection devices (wetted-wall cyclones, air-to-air concentrators, etc.) described in Chapter 4 must convert the air sample to a fluid (hydrosol) and provide it to the assay instrumentation.

For nucleic acid-based detection and identification technologies, one can expect some trade-off between the time required for detection and the starting concentration. Rugged, field-tested aerosol collectors such as the XM-2 have been demonstrated to capture particles in the 1 to 10 μm range with an efficiency of 50 percent or higher, with an effective concentration factor on the order of 5 × 105 (that is, one captured particle per 500 liters of air is concentrated into 1 milliliter of collection fluid.) Bioaerosol collectors of even higher performance have been developed under the guidance of the U.S. Army Edgewood Chem-Bio Center (ECBC) at Aberdeen Proving Grounds.5

From the standpoint of system analysis for the detect-to-warn application, some miniaturization of the overall system dimensions will decrease the time required to transport and prepare the samples for their detection assays, so collection into 1 milliliter is probably less desirable than collection into a 100 microliter or smaller liquid volume. It is important to remember that decreasing the size of the system may lead to more rapid clogging of the fluidics, so that shorter maintenance intervals may be required with the reduction in system size. The same considerations apply to the use of multistage precollection fractionators that could provide concentration factors exceeding 106; improved overall performance is likely to come at the expense of more frequent maintenance.

SAMPLE PREPARATION

Nucleic acid amplification and analysis methods are sensitive to contamination by inhibitors, which can often accompany samples that are collected from the open environment. Furthermore, the nucleic acid of interest typically resides within a cell or spore and must be liberated or made available to the other chemical components of the assay. (While it is true that there is often some detectable, exogenous DNA that was trapped on or within the exosporium during the process of sporulation, assays based solely on this source of DNA may be less sensitive and less reliable.) For reliable assays based on nucleic acid sequences, other than assays that are intracellular, the nucleic acid must be separated from interfering or inhibiting components before the assay is performed.

5  

E. Stuebing, U.S. Army. Discussions with the committee in 2003.



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